title: Phosphoproteome profiling using a fluorescent phosphosensor dye in two-dimensional polyacrylamide gel electrophoresis.
abstract: We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4-5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS.
authors.affiliation: Department of Life Sciences Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe, 650-8586, Japan.
authors.forename: Mieko
authors.initials: M
authors.lastname: Otani
authors.forename: Taizo
authors.initials: T
authors.lastname: Taniguchi
authors.forename: Akiko
authors.initials: A
authors.lastname: Sakai
authors.forename: Jouji
authors.initials: J
authors.lastname: Seta
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kadoyama
authors.forename: Tooru
authors.initials: T
authors.lastname: Nakamura-Hirota
authors.forename: Shogo
authors.initials: S
authors.lastname: Matsuyama
authors.forename: Keiji
authors.initials: K
authors.lastname: Sano
authors.forename: Masaoki
authors.initials: M
authors.lastname: Takano
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005456:Fluorescent Dyes; D010750:Phosphoproteins